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United States Department of Agriculture

Agricultural Research Service

Research Project: USING FUNCTIONAL AND APPLIED GENOMICS TO IMPROVE STRESS AND DISEASE RESISTANCE IN FRUIT TREES

Location: Appalachian Fruit Research Laboratory: Innovative Fruit Production, Improvement and Protection

Title: Transgenic Expression of the Erwinia Amylovora (Fire Blight) Effector Protein Eop1 Supresses Host Basal Defense Mechanisms in Malus (Apple)

Authors
item Lalli, Donna
item Artlip, Timothy
item Wisniewski, Michael
item Norelli, John (jay)

Submitted to: Mid Atlantic Plant Molecular Biology Society Conference
Publication Type: Abstract Only
Publication Acceptance Date: August 13, 2008
Publication Date: August 21, 2008
Citation: Lalli, D., Artlip, T.S., Wisniewski, M.E., Norelli, J.L. 2008. Transgenic expression of the erwinia amylovora (fire blight) effector protein eop1 supresses host basal defense mechanisms in malus (apple) . Mid Atlantic Plant Molecular Biology Society Conference. Page 32 in MAPMBS Program Book.

Technical Abstract: Erwinia amylovora (Ea) is the causative agent of fire blight, a devastating disease of apple and pear. Like many other plant and animal bacterial pathogens Ea utilizes a type three secretion system (TTSS) to deliver effector proteins into plant host cells. Once inside the host cell, effector proteins are thought to function as inducers and/or suppressors of host defense responses; however, the exact mechanisms by which Ea effectors regulate these responses are not clearly defined. Several Ea effectors have been identified based on their sequence similarity to both plant and animal bacterial pathogen effectors. Eop1 (Erwinia outer protein 1) has sequence similarity to the animal pathogen Yersinia pseudotuberculosis effector YopJ. To investigate the role of Eop1, M.26 apple was transgenically engineered to express the Ea effector protein Eop1 under the control of an inducible promoter. The Eop1 effector protein was directionally cloned from E. amylovora strain Ea273 into a Gateway (TM) compatible entry vector using gene specific primers that were modified to incorporate a Kozak sequence in the 5’ end of the effector gene to facilitate proper translation in a eukaryotic system and a 6His tag at the 3’ end for protein detection. Eop1 was subsequently cloned through Gateway technology into a binary vector, pBinPlusARS.XVE. This binary vector incorporates the regulatory elements of the estradiol-induced XVE gene expression system developed by Zuo et al. (2000) and was used in Agrobacterium-mediated transformation of apple. Transgenic apple lines were confirmed through PCR analysis with effector specific primers and evaluated for Agrobacteria contamination with virG specific primers. Inducible expression of eop1 in the presence of 25 uM estradiol was confirmed by RT-PCR. Callose deposition is a cellular marker of host basal defense, also known as innate immunity. When non-induced and induced leaf tissue of T98, a non-leaky Eop1 apple transgenic, was challenged with an Ea TTSS- mutant a significant reduction in callose deposition was observed in induced tissue, indicating that transgenic expression of Eop1 suppresses host basal defense mechanisms. In the future, these transgenic lines will enable us to continue investigating the role of Eop1 in regulating host defense mechanisms, as well as, determining its functionality when expressed in planta and its effect on host gene expression.

Last Modified: 4/19/2014
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